Synthesis , Antitumor and Antimicrobial Activities of Some Novel 1-( Substituted )-3-Methyl-1 H-Pyrazol-5 ( 4 H )-One

Several 1-(substituted)-3-methyl-1H-pyrazol-5(4H)-one as RA1-RA9 were synthesized and compounds were screened for antitumor activity against Ehrlich ascites carcinoma (EAC) cells and antimicrobial activity. Elemental analysis, mass spectral data, H-NMR, and IR confirmed the structure of the newly synthesized compounds. Some of the tested compounds showed good antitumor and antimicrobial activity. Compounds RA1, RA4, and RA9 exhibit highest antitumor activity against EAC cells in comparison with 5-flurouracil as standard drug.


Experimental
Analytical TLC was performed on Silica gel F254 plates (Merck) with visualization by UV light.All the products obtained were purified by column chromatography using silica gel (100-200 mesh).Hexane was used as a co-eluent.

Antitumor Activity
In vitro anticancer activity of compounds RA1-RA9 against EAC cells was determined by trypan blue exclusion method [16].The EAC cells were collected, counted and adjusted to 106 cells/mL with normal saline.The drug dilutions were made with phosphate buffer saline (PBS) and were further adjusted to concentrations ranging from 125-1000 µg/mL.The drug dilutions were then added to the EAC cells and incubated at 37 °C for 3 h.At the end of 3 h, the cell viability was determined and percentage cytotoxicity was calculated.
The percentage cytotoxicity was calculated using the formula: where, T c = total EAC cells and D c = dead EAC cells [17].
Compounds RA1-RA9 with significant in vitro anticancer activity were further selected for screening in vivo anticancer activity by determining different parameters like body weight analysis, mean survival time (MST) and percentage increase in life span (% ILS) [18].The EAC cells containing 106 cells/0.1 mL of phosphate buffer saline were injected into the peritoneal cavity of all the animals (six swiss albino mice in each group).Treatment with test compounds (90 mg/kg body weight) and the standard 5-flurouracil (520 µg/kg body weight) was started 24 h after inoculation of cancer cells, once daily as a single dose in 0.3% CMC suspension by intraperitoneal route for 10 days.All the mice were weighed daily up to 11 days.The decrease in body weight and MST (Mean Survival Time) of the test and standard group animals were compared with control group.Results are shown in Table 1.Percentage decrease in the body weight was determined by using the formula, percentage decrease in body weight = (G c -G t ) / G c × 100, where G c = gain in body weight of control group and G t = gain in body weight of treated group.Percentage increase in life span was calculated by the formula, % ILS = (MST of treated group -MST of control group) / MST of control group × 100.Student t-test was performed to ascertain the significance of the exhibited activity.

Hematological analysis [17]
In order to detect the influence of RA1-RA9 on hematological status of EAC-bearing mice, a comparison was made among four groups (n = 6) of mice on the 13 th day after tumor inoculation.Group I comprised of normal mice, group II comprised of EAC bearing mice, group III comprising EAC bearing mice treated with (RA1-RA9) (90 mg/kg/day for 10 days), and group IV having EAC bearing mice treated with 5-flurouracil (520 μg/kg for 10 days).Blood was drawn from each mouse by the retro orbital plexus method and the white blood cell count (WBC), red blood cell count (RBC), hemoglobin and percentage differential count were determined.The results are given in Table 2.

Antibacterial and antifungal activities
Applying the agar plate diffusion technique [19] all of the newly synthesized compounds were screened in vitro for antibacterial activity against Escherichia coli (E.coli), Pseudomonas aeruginosa (Gram-negative), Staphylococcus aureus, Bacillus subtilis (Gram-positive) at 20 μg/ml, 30 μg/ml, 40 μg/ml concentrations, respectively.Under identical conditions, the positive control antibiotics Amoxicillin at 40 μg/ml showed zone of inhibition 26-24 mm and 22-25 mm for Gram-negative and Gram-positive organism respectively.Similarly, the antifungal screening of the compounds was carried out against fungi C. albicans and A. niger by using fluconazole (40 mg/ml) as the positive control, which showed 27 mm and 28 mm, respectively, as the zone of inhibition.

Results and Discussion
In vivo antitumor screening reveals that some of the tested compounds are promising candidates having good activity against EAC cells.Compounds RA1, RA4, and RA9 exhibit highest antitumor activity than standard antitumor agent.Compounds RA2, RA3, RA5 exhibit nearly same antitumor activity comparable to standard.Compounds RA6, RA7, RA8 are inactive against EAC cells.Antimicrobial study reveals that compounds RA1, RA4, RA6 and RA9 exhibit good antibacterial and antifungal activities (Tables 3  and 4).Compound RA9 exhibited the highest degree of antimicrobial activity than standard drugs.Rest of the compounds does not show any significant antimicrobial activity as compared with standard agent.Structure activity studies of the title compounds for antitumor activity reveal the importance of N-1 position of pyrazolone ring as it should contain N-methyl substitution followed by ethyl linkage as RA1 (144.68),RA4 (153.90),RA9 (165.95) increases the percentage increases in life span (%ILS) as compared to compound RA6 (24.32),RA7 (11.34),RA8 (16.95) having heterocyclic substitution at N-1 position followed by ethyl linkage.
Hematological parameters of EAC bearing mice on day 13 showed significant changes when compared to normal mice (Table 2).The total WBC count was found to increase with a reduction in the hemoglobin content of RBC.The differential count of WBC showed that the percentage of neutrophils increased while that of lymphocytes decreased.At the same time treatment with compounds RA1, RA4, and RA9 could change these altered parameters to near normal.

Synthesis of 3-methyl-1H-pyrazol-5(4H)-one (1): This
1H NMR was recorded in Brucker 400 MHz spectrometer.LC-MS was used for the mass spectral analysis.IR spectra were recorded on a FT-IR spectrometer using KBr pellets.Elemental analysis was carried out in CHN analyzer EA-1112, Thermo Finnigan.All spectral studies were carried out at elemental analysis laboratory in the School of Chemistry, University of Hyderabad, India.Melting points were determined in open capillaries on a Thermonik melting point apparatus, Mumbai, India and are uncorrected.
a = Zone of inhibition in millimeter.
a = Zone of inhibition in millimeter.